The Sanger chain-termination sequencing method and the Polymerase Chain Reaction (PCR) are two powerful methods for analyzing DNA which are certain to become even more widely used in the near future. These procedures both use the template-directed extension of an oligonucleotide primer by a DNA polymerase followed by the analysis of the primer extension products, most often by gel electrophoresis. The analysis of these extension products is often complicated by the presence of the other reaction components which comprise the bulk of the mixture and which are difficult and tedious to remove. Thus, both methods would benefit from a simplified procedure for the isolation of these primer extension products from other reaction components and for the generation of them in a form appropriate for gel electrophoresis. In addition to Sanger sequencing and PCR, other primer extension reactions are being developed which will benefit from such a procedure.
The worldwide effort to sequence the human genome will require many thousands of Sanger sequencing reactions. By using fluorescence detection, the GENESIS 2000.TM. (E. I. du Pont de Nemours and Co., Wilmington, Del.) greatly simplifies and automates the electrophoretic analysis of Sanger sequencing reactions. However, the work-up of these reactions requires the use of spin columns or precipitations to remove unincorporated fluorescent terminators followed by the evaporation of a large volume of eluting solvent. Since processing a large number of sequencing reactions is very tedious, a faster, simplified work-up procedure is highly desirable. Standard radioisotope-based Sanger sequencing reactions of plasmid DNA normally do not require any work-up prior to gel electrophoresis. However, sequencing reactions of more complex DNA such as cosmid, lambda clone, or genomic DNA generate primer extension products contaminated with large amounts of template DNA and/or extraneous labeled DNA fragments which tend to interfere with gel electrophoresis. Sequencing of complex DNA would benefit from a simple procedure for isolation of the labeled extension products from the other reaction components.
The Polymerase Chain Reaction (PCR) is likely to become integral to the new field of DNA diagnostics. The technique generates two amplified complementary strands of DNA in the presence of double-stranded template. By means of multiple primer extension reactions, millions of double stranded copies of a specific region of a template that lies between two primers are produced. Analysis of the amplified DNA (for example by sequencing or direct gel electrophoresis) is best performed on a single strand of DNA uncontaminated with the complementary strand or the template. Analysis would be facilitated by the availability of a simple procedure for the isolation of the extension products from the other reaction components.
The biotin-avidin (streptavidin) system is a very useful analytical tool and is utilized in a wide variety of bioanalytical applications. The proteins avidin and streptavidin (hereinafter referred to jointly as "strept/avidin") form exceptionally tight complexes with biotin (K.sub.D =10.sup.-15 M) and certain analogs of biotin. In general, when biotin is coupled to a second large or small molecule through its carboxyl side chain, the resulting conjugate is still tightly bound by strept/avidin. The second molecule is said to be "biotinylated" when such conjugates are prepared. The biotin-strept/avidin binding pair is utilized in a wide variety of bioanalytical applications. These applications generally involve complexation of a biotinylated analyte to strept/avidin followed by detection, analysis, or use of the complex. For a review of this field, see Wilchek et al. (Anal. Biochem., 171, 1-32, 1988). In a few cases, the complex between the biotinylated analyte and strept/avidin is disassociated before the analysis is complete. The simple complex between biotin and avidin can be disassociated by: heating at 132.degree. C. (but it reforms on cooling) or denaturation with 6M guanidine hydrochloride at low pH. Due to the harshness of these conditions, complexation of a biotinylated analyte can be considered to be effectively an irreversible process. Such dissociation conditions are likely to destroy many biological analytes.
The biotin-strept/avidin complex has been used frequently in the analysis of biotinylated nucleic acids. However, there are only three reports concerning the disassociation of such complexes.
Disclosures involving biotinylated nucleic acids include the misinterpretation by Mitchell et al., (Anal. Biochem., 178, 1-4, 1989) of Delius et at. (Nucleic Acids Res., 13, 5457-5469, 1988) that biotinylated single-stranded DNA fragments could be dissociated from avidin-agarose by 50% guanidine isothiocynate/formamide at room temperature. In fact, Delius et al., disclose a method that separates complementary strands of biotinylated DNA, but does not dissociate the biotinavidin complex from the DNA strands with formamide. Other disclosures include the report by Richardson et al. (Nucleic Acids Res., 11, 6167-6184, 1983) that a biotinylated ribonucleotide trimer could be eluted from an avidin-agarose column with a large quantity of 6M guanidine hydrochloride (pH 2.5); the report by Eckermann et al. (European J. of Biochem., 82, 225-234, 1978) that the complex between avidin and biotinylated ribosomal RNA could be disrupted by treatment with 70% formic acid for 10 minutes at room temperature.
In the above three disclosures, the biotinylated nucleic acids released from strept/avidin were not carefully analyzed to prove that the released nucleic acid was unmodified and that all of the binding protein had been removed from the biotin subunit. In fact, Delius asserts that the biotinylated nucleic acid eluted from a solid-supported avidin was still complexed to some of the binding protein. Treatment of nucleic acids with acid in general, and formic acid in particular, is known to cause depurination and eventually strand cleavage. Therefore complex dissociation under the above conditions might be expected to release modified nucleic acid for analysis. The most common and sensitive method for analyzing nucleic acids is gel electrophoresis, but there are serious obstacles to analyzing nucleic acids decomplexed as described above. Both 6M guanidine hydrochloride (pH 2.5) and 70% formic acid are likely to be incompatable with gel electrophoresis. Fifty percent guanidine isothiocyanate in formamide is also a less than optimum choice since samples with high salt content tend to produce poorly resolved electrophoresis bands. The use of any of these three methods to dissociate biotinylated sequencing fragments from solid supported avidin or streptavidin would likely require further treatment of the resulting solution of fragments before analysis by polyacrylamide gel electrophoresis. In conclusion, successful electrophoretic analysis of biotinylated nucleic acids disassociated from strept/avidin is problematical and has not been previously demonstrated.
A related disclosure by Shimkus et al. (Proc. Natl. Acad. Sci. USA, 82, 2593-2597, 1985) reports that DNA probes containing biotin attached through a chemically cleavable disulfide group bind to avidin-agarose columns and can be eluted from the column with aqueous dithiothreitol which breaks the disulfide bond, leaving the biotin-avidin complex on the column. The use of disulfide linkages is not preferred because many enzymes require the presence of thiols for activity. There are three techniques known in which 5'-biotinylated oligonucleotides are used as primers in template directed extension reactions; however, in none of these is the biotin-avidin complex broken nor can the biotinylated extension products be analyzed by gel electrophoresis. In one disclosure, Mitchell et al. (Anal. Biochem., 178, 1-4, 1989) describe a method for direct dideoxy sequencing following PCR in which the biotinylated extension product is captured by solid-supported streptavidin and the complementary strand is removed by base denaturation; however, the biotin-streptavidin bond is never broken, and it is the unbiotinylated complementary strand which is analyzed by dideoxy sequencing. In another disclosure, Landegren et al (Science, 241, 1077-1081, 1988) describe a method for ligase-mediated gene detection in which a 5'-biotinylated primer is ligated to a radioactively-labeled oligonucleotide in a template directed manner and the now labeled biotinylated strand is captured by solid-supported streptavidin beads; the beads are then analyzed for the presence of a label, again without breaking the biotin-streptavidin bond. In a third disclosure, Richterich (Nucleic Acids Res., 17, 2181-2186, 1989) describes a method for non-radioactive sequencing of DNA in which a 5'-biotinylated primer is used in a Sanger sequencing reaction, not to isolate the sequencing fragments but only to detect them.
Hultman et al (Nucleic Acids Res., 17, 4937-4946, 1989) disclose a method for direct solid phase sequencing of genomic and plasmid DNA using ferromagnetic beads as a support. In this sequencing procedure the template (not the fragments) is biotinylated and attached to streptavidin Dynabeads.TM. (Dynal, Inc.). Again, the resulting biotin-streptavidin bond is never broken.